Alcoholism is a serious health and socioeconomic problem in the U.S. Understanding how alcohol produces reward, motivates further consumption, and eventually leads to addiction is necessary to design effective treatments for alcohol use disorders (AUDs). Synaptic transmission mediates all of these behaviors, however, little is known about the effect of alcohol on synaptic transmission in the context of human neurons. The single nucleotide polymorphism (SNP) rs1799971 (OPRM1 A118G) produces a non-synonymous amino acid substitution in the mu-opioid receptor (MOR), in which Asparagine 40 (MOR N40) is replaced with Aspartate (MOR D40), and is associated with AUDs in specific ethnic groups. Importantly, Naltrexone, a nonselective MOR antagonist, has potent therapeutic effects in alcoholic individuals with MOR D40. We generated human neurons from 7 subjects carrying either homozygous MOR N40 or MOR D40. Our preliminary data suggest that human neurons carrying D40 show defective re-sensitization after MOR activation by DAMGO ([D-Ala2, NMe-Phe4, Gly-ol5]-enkephalin), suggesting defective trafficking of MORs. Supporting this, bioinformatic analyses and mouse models of human MOR N40D suggest that D40 disrupts an N-glycosylation site on MOR. However, the mechanism by which MOR protein trafficking defects affect the interaction between ethanol and opioids is not known. It is particularly important to reveal the molecular mechanisms underlying the function of N40D MOR variants in their native neuronal context because previous studies performed in heterologous systems have revealed inconclusive and confusing results. Moreover, a species-specific trafficking mechanism of MORs has been suggested. The objective of this proposal is to study the impact of alcohol and opioid signaling in both mouse and human neurons carrying both the N40 and D40 MOR allelic variants, focusing on the synaptic mechanisms that likely underlie behavior. The central hypothesis is that defective D40 MOR trafficking results in an altered effect of the interaction between alcohol and opioids on synaptic function in the reward neurocircuitry. We will first examine the effect of alcohol on synaptic function in a defined neurocircuitry composed of human neurons carrying these gene variants. Next, we will use a mouse model of human N40D, and study the synaptic mechanism in the reward neurocircuitry, i.e. the midbrain ventral tegmental area (VTA), in relation to MOR function and ethanol. The proposed research is innovative, because we will combine recent developments in stem cell biology, the state-of-the-art synaptic physiology, and novel microfabrication technologies to directly probe the impact of alcohol and opioid signaling on synaptic function in both mouse and human neuronal networks carrying OPRM1 gene variants. We expect to unravel a species- and cell type-specific mechanism of MOR N40D variants that may provide novel information for understanding AUDs.